In late 1997, we initiated a study to identify minimal levels of HIV-1 infection in seronegative persons who reported repeated high-risk sexual exposures to HIV-1-infected partners, termed as exposed seronegative (ES). Among ten well-defined ES individuals with detectable HIV-1-specific cytotoxic T-lymphocytes (CTL) responses, we demonstrated presence of HIV-1 infection in two ES (Appendix A) at extraordinarily low levels (range: 0.1 - 0.01 copies per million cells) that are well below the detection limit of conventional assays. Using same methodologies, we identified parallel evidence for low levels of SIV infection in macaques that were transiently viremic or vaccine-protected by conventional testing. Most recently, we have found persistent low levels of HIV-1 infection in three vaccinated persons who were transiently viremic by conventional assays. These findings indicate that minimal levels of HIV-1 infection (MLHI) can be demonstrated in ES and vaccinated persons, only by improved technologies with hundreds or thousands of PCR amplifications and sequence analyses on large amounts of cells. Here, we propose to extend our preliminary results to mechanize the processes and to assess for MLHI more rigorously among our cohort of ES and vaccine recipients from NIH-funded vaccine trials, mainly the HIV Vaccine Trails Network (HVTN). We will also take advantage of the availability of virus or sequences isolated from our assays to characterize the genotype of virus in breakthrough MLHI infection and compare these characteristics with vaccine strains. The Specific Aims are: 1. To improve methodologies to identify transient or persistent minimal levels of HIV-1 infection in vaccine trial participants and ES individuals. We will ascertain the frequency and replication state of HIV-1 in persons who may have transient infection as indicated by conventional virologic, or serologic, or T cell-mediated immunologic assays. 2. To characterize the genotype of HIV-1 strains present in peripheral blood and tissues in MLHI persons and to determine if there is impact of vaccination or repeated exposures to HIV-1 on the attenuated minimal levels of HIV-1 infection. These studies should assist definition of virologic and immunologic characteristics required to attenuate and/or substantially control HIV-1 infection, a critical goal of AIDS vaccine development.